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Treatment of chronic allodynia in spinally injured rats: effects of intrathecal selective opioid receptor agonists
Pain 75 (1998) 209–217
Treatment of chronic allodynia in spinally injured rats: effects of intrathecal selective opioid receptor agonists Jing-Xia Hao, Wei Yu, Zsuzsanna Wiesenfeld-Hallin, Xiao-Jun Xu* Department of Medical Laboratory Sciences and Technology, Division of Clinical Neurophysiology, Karolinska Institute, Huddinge University Hospital, S-141 86 Huddinge, Sweden Received 26 August 1997; received in revised form 20 November 1997; accepted 4 December 1997
Abstract We examined the effects of intrathecal (i.t.) selective opioid receptor agonists in alleviating mechanical and cold allodynia in spinally injured rats. Both DAMGO ([D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin, a m-opioid receptor agonist) and DPDPE ([D-Phe2,D-Phe5]-enkephalin, a d-opioid receptor agonist) dose-dependently relieved the chronic allodynia-like behavior at doses selective for their respective receptors. The anti-allodynic effect of DAMGO and DPDPE was reversed by the selective m- and d-opioid receptor antagonists CTOP (DPhe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2) and naltrindole, respectively. In contrast, the selective k-opioid receptor agonist U50488H did not alleviate the allodynia-like behavior, but rather enhanced it. The anti-nociceptive and anti-allodynic effect of i.t. DAMGO was blocked by U50488H. Thus, activation of spinal m- and d-, but not k-opioid receptors produced anti-allodynic effect in this model of central pain. Drugs which act selectively on opioid receptor subtypes may be useful in managing chronic central pain of spinal cord origin. 1998 International Association for the Study of Pain. Published by Elsevier Science B.V. Keywords: Chronic pain; Intrathecal; Morphine; Neuropathic pain; Opioids; Spinal cord injury
1. Introduction Neuropathic pain, which can occur after nervous system injury, is usually difficult to treat. This is particularly true for central pain after injury or dysfunction to the central nervous system (CNS) (Tasker, 1990; Boivie, 1994). The successful development of suitable animal models for these human pain conditions will not only be useful in elucidating the mechanisms of these painful states, but also in exploring effective treatments. Several animal models for neuropathic pain following peripheral nerve injury are available which have generated a large body of literature on the pharmacological treatment of the abnormal painrelated behaviors in these models (Bennett, 1993; Zeltser and Seltzer, 1994). We have recently developed a rat model of chronic pain-
* Corresponding author. Division of Clinical Neurophysiology, Huddinge University Hospital, S-141 86 Huddinge, Sweden. Tel.: +46 8 58582213; fax: +46 8 7748856; e-mail: [email protected]
related behaviors after ischemic spinal cord injury which in many aspects mimics central pain in spinally injured patients. Thus, we have observed marked mechanical and cold allodynia-like behaviors in spinally injured rats in the dermatomes at or rostral to the injured spinal segments (Xu et al., 1992a, 1994). Furthermore, animals also exhibited autotomy and ex-cessive scratching, behaviors that may indicate the presence of spontaneous pain or dysesthesia (Xu et al., 1992a). We and others have suggested that this may represent an animal model for central pain of spinal origin which provides a valuable tool to study the mechanisms and treatments of this particular type of refractory painful condition (Yezierski, 1996; Wiesenfeld-Hallin et al., 1997). We have conducted systematic studies examining the pharmacological management of chronic allodynia-like behaviors in these spinally injured rats. Similar to some clinical reports, we found that systemically administered local anesthetics, tocainide and mexiletine, were effective in alleviating the chronic allodynia-like behavior (Xu et al., 1992a,b). Furthermore, the allodynia could be relieved by
0304-3959/98/$19.00 1998 International Association for the Study of Pain. Published by Elsevier Science B.V. PII S0304-3959 (97 )0 0221-2
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systemic administration of antagonists of N-methyl-daspartate (NMDA) receptors, nitric oxide synthase inhibitors and antagonists of CCKB receptors (Xu et al., 1994; Hao and Xu, 1996a,b), indicating the possible involvement of these transmitter systems in the generation and maintenance of the chronic allodynia-like state. We are particularly interested in exploring the anti-allodynic efficacy of morphine in our model of central pain as it is a controversial issue in clinic practice whether or not opiates are effective in treating neuropathic pain (Arne´r and Meyerson, 1988; Arne´r and Meyerson, 1993; Portenoy et al., 1990; McQuay et al., 1992). Moreover, the efficacy of morphine in treating central pain has not been thoroughly evaluated (Hammond, 1991), although the general impression is that morphine is rather ineffective (Boivie, 1994; Bowsher and Nurmikko, 1996). We have evaluated the efficacy and potency of morphine in alleviating the allodynialike behaviors in this model after systemic, intrathecal (i.t.) and intracerebroventricular (i.c.v.) administration (Yu et al., 1997a). Systemic morphine was ineffective in alleviating allodynia at sub-sedative doses. Similarly, i.c.v. morphine only reduced the vocalization component of the allodynic responses. In contrast, i.t. morphine dose-dependently alleviated allodynia without causing sedation, although the doses of morphine required to produce the anti-allodynic effect were higher than those for anti-nociception. These results suggest that spinal administration of morphine may be useful in treating central pain in patients with spinal cord injury (Yu et al., 1997a). Three major receptor subtypes, m, d and k, mediate the actions of opioids in the nervous system (Martin et al., 1976; Yaksh, 1987; Mansour et al., 1988). Agonists of all three opioid receptors induce anti-nociception at the spinal level (Yaksh, 1987), although the effects of k-opioid receptor agonists are less clear (Schmauss and Yaksh, 1984; Knox and Dickenson, 1987; Millan, 1989; Hylden et al., 1991). It has also been reported that spinal administration of k-opioid receptor agonists antagonized the effects of morphine and other m-opioid receptor agonists (Dickenson and Knox, 1987; Fujimoto et al., 1990; Song and Takemori, 1991). The present studies were undertaken to explore the effects of i.t. administration of selective agonists for m-, d- and k-opioid receptors on the mechanical and cold allodynia-like behaviors in spinally injured rats. The selective antagonists CTOP (Kramer et al., 1989) and naltrindole (Portoghese et al., 1988) were used to further confirm the receptor subtypes involved. The interactions between the k-opioid receptor agonist U50488H and mopioid receptor agonist DAMGO were also examined.
2. Materials and methods 2.1. Photochemically-induced spinal cord ischemia The subjects were female Sprague–Dawley rats (B & K Universal, Stockholm, Sweden). All experiments were
approved by the local research ethics committee. The method for inducing photochemical spinal cord lesion has been described in detail previously (Xu et al., 1992a). Briefly, the rats were anesthetized with chloral hydrate (Sigma; 300 mg/kg, i.p.) and one jugular vein was catheterized. Vertebrae T11–L2 were exposed after a midline incision of the skin on the back. The animals were positioned beneath a tuneable argon ion laser (Innova, Model 70, Coherent Laser Products Division) and irradiated with a knife edge beam, which was used to cover the T13 vertebra, with an average power of 0.16 W for 10 min. No laminectomy was performed. Immediately before and 5 min after the start of the irradiation, erythrosin B (Red No. 3, AldrichChemie) was injected intravenously in 0.9% saline at a dose of 32.5 mg/kg. After irradiation the incision was closed and the animals were kept warm for 2 h. Their bladders were emptied manually 2–3 times a day until normal function was regained. 2.2. Test of responses to mechanical and cold stimuli A set of calibrated von Frey hairs was used to test the vocalization thresholds of the rats to graded mechanical touch/pressure ranging from 0.021 to 410 g. During testing the rats were gently restrained in a standing position and the von Frey hair was pushed onto the skin until the filament became bent. The frequency of the stimulation was about 1 Hz and at each intensity, the stimuli were applied 5–10 times. The intensity of stimulation which induced consistent vocalization (.75% response rate) was considered as pain threshold. Although a number of reactions were noted in response to light mechanical stimulation in allodynic rats after spinal cord lesion (such as biting, escaping and hindlimb flexion), vocalization was chosen to represent the pain-like response as it was most consistent and easiest to characterize. The responses of rats to tactile stimulation was tested with the blunt point of a pencil gently brushing the skin of the rats in a rostral-caudal direction. The frequency of the stimulation was about 1 Hz and responses were graded with a score of 0, no observable response; 1, transient vocalization and moderate effort to avoid probe; 2, consistent vocalization and aversive reactions; and 3, sustained and prolonged vocalizations, aggressive behaviors. Normal rats exhibit no reactions to such tactile stimuli (score 0). The response of the rats to cold stimuli was evaluated by spraying ethyl chloride (Medikema AB, Perstorp, Sweden) on the shaved skin area exhibiting mechanical allodynia. The response of the rat was graded with a score of 0, no observable response; 1, localized response (skin twitch and contraction), no vocalization; 2, transient vocalization, moderate struggle; and 3, sustained vocalization and aversive reactions. The spray produced an intense cold sensation when applied to the experimenter’s forearm and skin temperature was reduced to around 0°C immediately after application, with rapid recovery.
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The light beam from a projector bulb was focused 1–2 cm from the tip of the tail and the latency of the tail flick was measured. The intensity of the thermal stimulus was adjusted so that the baseline latency was at 3–4 s and a 10 s cut off time was imposed to avoid tissue damage.
groups. The data are expressed as medians ± MAD (median derived absolute value) or as mean ± SEM and were analysed with the Wilcoxon signed-ranks test. In order to estimate ED50 and compare the anti-allodynic and antinociceptive potency of DAMGO and DPDPE, we have also calculated the % maximal possible effect (MPE) based on the formula:
2.4. Implantation of i.t. catheters
%MPE = (post − drug response − baseline=
2.3. Tail flick test
The i.t. catheter was implanted according to a modification of the technique described by Storkson et al. (1996). After anesthesia with methohexital (Brietal, Lilly, IN, USA), a catheter (PE 10, o.d. 0.61 mm) was inserted into the subarachnoid space through a guide cannula connected to a 20 gauge needle which punctured the dura at the level of the cauda equina. The catheter was then carefully implanted rostrally, aiming its tip at the lumbar enlargement. The proper location of the catheter was examined before the actual pharmacological experiments by assessing sensory and motor blockade after i.t. injection of 7 ml lidocaine (50 mg/ml; Astra, So¨derta¨lje, Sweden) and was confirmed after the experiments by laminectomy. 2.5. Experimental design Thirteen chronic allodynic rats were randomly assigned to three groups and DAMGO (n = 6), DPDPE (n = 4) or U50488H (n = 3) were administered i.t. in a cumulative dose regime at 20 min intervals. The von Frey hair thresholds and the responses of the rats to tactile and cold stimulation were assessed before and every 10 min after i.t. administration. The tail flick latency was only tested once after each dose at 10 min in order to avoid tissue damage. The rats that received DAMGO as their first injection were used again 1 week later where DPDPE (n = 4) or U50488H (n = 2) were administered. No differences in the effects of DPDPE and U50488 were noted between rats that did or did not receive DAMGO previously and thus results from these two experiments were combined for data presentation and analysis. 2.6. Drugs and statistics DAMGO [(D-Ala2,N-Me-Phe4,Gly5-ol)-enkephalin], DPDPE [(D-Phe2,D-Phe5)-enkephalin] and CTOP (D-PheCys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2) were obtained from Peninsula Laboratories (Merseyside, UK). U50488H (trans-(±)-3,4-dichloro-N-methyl-N-[2-(1-pyrolidinyl)cyclohexnoyl]benzeneacetamide methanesulphonate) and naltrindole (17-cyclopropylmethyl-6,7-dehydro-4,5a-epoxy-3,14-dihydro-6,7-2′,3′-indolomorphinan) were from Research Biochem (Natick, MA, USA). All drugs were dissolved in saline and injected i.t. in a volume of 10 ml followed by 15 ml saline to flush the catheter. Rats were randomly assigned to the different treatment
maximal response − baseline) × 100 where maximal response is 10 s for the tail flick test and 73 g (response threshold in normal rats) in the von Frey hair test, respectively. Linear regression was used to examine the dose-dependent effect of DAMGO and DPDPE and to calculate ED50.
3. Results 3.1. Allodynia-like behaviors in spinally injured rats Two weeks to 3 months after the ischemic injury, mechanical and cold allodynia-like responses were observed in about 40% of the rats (Xu et al., 1992a; Hao and Xu, 1996a; Hao and Xu, 1996b). The vocalization threshold to pressure exerted by von Frey hairs in normal rats was 73– 95 g and there were no reactions to subthreshold stimuli. In contrast, there was a marked decrease in vocalization threshold to mechanical pressure (median 0.94 g, range 0.18–7.1 g) in the animals with pain-related behavior. Other behaviors, such as skin twitch, jumping and escaping was also evoked by low intensity mechanical touch/pressure. Light tactile stimulation triggered painful reactions in some, but not all, rats where low intensity mechanical touch/pressure generated painful reactions. In response to the cold spray, normal rats exhibited no or local responses (score 0 or 1). In contrast, the allodynic rats exhibited clearcut aversive reactions, including vocalization, licking or scratching of the stimulated area, avoidance and escaping. Mechanical and cold allodynia-like behaviors were usually found in the same animal. The skin areas where increased response to mechanical and cold stimulation could be observed corresponded to one or more dermatomes at the rostral edge of the lesioned spinal segment. As the epicenter of the lesion was near the lumbar enlargement, the allodynic area was usually at the mid or lower back and abdomen. In the majority of rats the symptoms lasted for months without signs of remission. The pain-like behaviors occurred only when the rats were tested and the animals otherwise exhibited no overt signs of discomfort. Rats that were included in the present study were irradiated 3–8 months prior to the pharmacological experiments, and all exhibited robust allodynia-like behavior to mechanical and cold stimuli for at least 1 month. Some rats
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also exhibited tactile allodynia. Some motor deficits were observed in these rats, which were mainly expressed as minor deficits in walking, slowed righting reflexes and reduced performance on the inclined plane test. 3.2. Effect of DAMGO The smallest dose of DAMGO tested (1 ng) produced no significant effect on vocalization threshold to von Frey hair stimulation (Fig. 1A), tail flick latency (Fig. 1B) and cold responses (Fig. 2A). A cumulative dose of 10 ng DAMGO increased the vocalization threshold of four out of six rats to stimulation with von Frey hairs. In fact the response thresh-
old was the same as in normal animals (73 g) (Fig. 1A). The responses of five out of six rats to cold was also normalized (Fig. 2A). The tail flick latency was also significantly prolonged by DAMGO at this dose (Fig. 1B). At a 30 ng cumulative dose, DAMGO normalized the vocalization threshold of all rats (Fig. 1A) and the tail flick latency reached cut-off value in five out of six rats (Fig. 1B). Interestingly, at this dose, the response of all rats to cold was abolished (score 0) which is below normal response. No further increase in vocalization threshold to stimulation with von Frey hairs was observed after a 100 ng cumulative dose of DAMGO, with only two out of six rats showing a response threshold (103 g) that was somewhat higher than normal values (Fig. 1A). Four of the six rats also exhibited tactile allodynia (Fig. 2B). These responses were also dose-dependently reduced by i.t. DAMGO in a fashion similar to the changes in the vocalization threshold to stimulation with von Frey hairs (Fig. 2B). In three rats, the duration of action of 100 ng i.t. DAMGO was followed and its anti-allodynic and anti-nociceptive effect was maintained for 45 min, partially recovered at 60 min and fully reversed at 90 min. In another three rats, the selective m-opioid receptor antagonist CTOP (10 or 30 ng) was administered i.t. 20 min after a cumulative dose of 100 ng DAMGO and a rapid reversal of the anti-allodynic and anti-nociceptive effect of DAMGO was observed. 3.3. Effect of DPDPE
Fig. 1. Effects of cumulative doses of intrathecal (i.t.) DAMGO on vocalization threshold to mechanical touch/pressure (A) and tail flick latency (B) in allodynic rats (n = 6). Note the logarithmic scale on the x-axis in both (A) and (B) and on the y-axis in (A). In this figure and Figs. 3 and 5, data are expressed as median ± MAD for vocalization threshold and as mean ± SEM for tail flick latency. DAMGO caused significant increase in vocalization threshold and tail flick latency starting from a 10 ng cumulative i.t. dose (*P , 0.05, compared to control value with Wilcoxon signed-ranks test).
Intrathecal DPDPE at 1 mg produced marked increase in vocalization threshold to von Frey hair stimulation in four out of eight rats. However, in the remaining four rats, there was little or no effect (Fig. 3A). DPDPE did not produce a significant effect on the response to cold at 1 mg (Fig. 4A). Increasing the dose of DPDPE to 3 mg markedly increases the vocalization threshold in all rats tested with normalization observed in five out of eight rats (Fig. 3A). The response to cold was also significantly reduced at this dose (Fig. 4A). At a cumulative dose of 10 mg DPDPE, the vocalization threshold to von Frey hairs and cold responses were normalized in all rats (Figs. 3A and 4A). No further increase in vocalization threshold or decrease in cold response was noted after a cumulative dose of 30 mg DPDPE (Figs. 3A and 4A). Four of the eight rats tested also exhibited tactile allodynia. These responses were dose-dependently reduced by i.t. DPDPE in a fashion similar to changes in vocalization threshold to von Frey hairs (Fig. 4B). The tail flick latency was also dose-dependently prolonged by i.t. DPDPE with a significant effect already observed at 1 mg (Fig. 3B). In four rats, the duration of action of a cumulative dose of 30 mg DPDPE was followed and was found to last between 30 and 45 min. In another four rats, the selective d-opioid receptor antagonist naltrindole (10 or 30 mg) was administered i.t. 10 min after 30 mg i.t. DPDPE and rapid reversal of
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threshold to von Frey hair stimulation was noted after a 100 ng cumulative dose of U50488H (Fig. 5A). Increasing the dose of U50488H to 1 or 10 mg still did not produce an anti-allodynic effect (Fig. 5A). Intrathecal U50488H also triggered tactile allodynia in rats which did not exhibit such behavior before the experiments and enhanced the already existing tactile allodynia (Fig. 6B). Intrathecal U50488H did not significantly alter the tail flick latency (Fig. 5B). 3.6. Effect of U50488H on the anti-allodynic effect of DAMGO One hundred nanograms DAMGO was administered i.t. 20 min after a 10 mg cumulative dose of U50488H in four rats. In contrast to the effectiveness of this large dose of DAMGO in suppressing the allodynia-like behavior in rats
Fig. 2. Effects of cumulative doses of i.t. DAMGO on responses of rats to cold (A) and tactile (B) stimulation. In this figure and Figs. 4 and 6, the number of rats showing different response scores are illustrated and the scores are represented as open columns (score 0); hatched columns (score 1), densely hatched columns (score 2) and dotted columns (score 3) (see Section 2 for detailed description of scoring criteria). The Wilcoxon signed-ranks test indicated that DAMGO caused significant reduction in the cold response starting at 10 ng (P , 0.05) and in the tactile response starting at 30 ng (P , 0.05).
the anti-allodynic and anti-nociceptive effect of the agonist was observed. 3.4. Comparison of the potency for the anti-allodynic and anti-nociceptive effect of DAMGO and DPDPE In order to compare the potency of DAMGO and DPDPE on reactions to different types of stimuli, the responses to von Frey hairs and tail flick test were converted to % change. Regression analysis indicated that both DAMGO and DPDPE caused significant dose-dependent anti-allodynic and anti-nociceptive effects. The ED50 for the anti-allodynic and anti-nociceptive effects of DAMGO were 5.8 and 12.7 ng, respectively and for DPDPE 0.53 and 0.34 mg, respectively. 3.5. Effect of U50488H U50488H administered i.t. at 1 or 10 ng did not significantly alter vocalization threshold to mechanical pressure and cold responses (Figs. 5A and 6A). However, at a 30 ng cumulative dose, there was a significant decrease in vocalization threshold to von Frey hair stimulation (Fig. 5A). There was also an increase in the cold responses after 30 ng U50488H (Fig. 6A). Further reduction in vocalization
Fig. 3. Effects of cumulative doses of i.t. DPDPE on vocalization threshold to mechanical touch/pressure (A) and tail flick latency (B) in allodynic rats (n = 8). Note the logarithmic scale on the x-axis in both (A) and (B) and on the y-axis in (A). DPDPE cause a significant dose-dependent increase in vocalization threshold and tail flick latency (*P , 0.05, compared to control value with Wilcoxon signed-ranks test).
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which did not receive U50488H previously, three of four rats failed to respond to DAMGO and only a moderate increase in vocalization threshold was observed in one rat. The anti-nociceptive effect of DAMGO was also reduced by U50488H.
4. Discussion 4.1. Anti-allodynic effect of m- and d-opioid receptor agonists In the present study, we observed that i.t. administration of selective agonists of m- or d-opioid receptors dose-dependently alleviated the mechanical allodynia-like behavior in spinally injured rats. The doses of these two agonists employed were comparable to or lower than those used in previous studies (Drower et al., 1991; Miaskowski et al.,
Fig. 5. Effects of cumulative doses of i.t. U50488H on vocalization threshold to mechanical touch/pressure (A) and tail flick latency (B) in allodynic rats (n = 5). Note the logarithmic scale on the x-axis in both (A) and (B) and on the y-axis in (A). U50488H caused a significant decrease in vocalization threshold (*P , 0.05, compared to control value with Wilcoxon signed-ranks test). No significant effect on tail flick latency was observed.
Fig. 4. Effects of cumulative doses of i.t. DPDPE on the responses of rats to cold (A) and tactile (B) stimulation. Wilcoxon signed-ranks test indicated that DAMGO caused significant reduction in cold response starting at 3 mg (P , 0.05). No significant effect on tactile response was observed due to the low number of rats exhibiting such behavior before DPDPE administration.
1991; Malmberg and Yaksh, 1992; Tiseo and Yaksh, 1993; Guirimand et al., 1994). The effects of the agonists were reversed by respective antagonists, indicating receptor mediation of the anti-allodynic effects of the m- and d-opioid agonists. No noticeable side effects, such as sedation or motor deficits, were observed after even the highest doses of these two opioid receptor agonists. In agreement with previous studies, i.t. DAMGO or DPDPE caused potent thermal anti-nociception (Drower et al., 1991; Malmberg and Yaksh, 1992; Tiseo and Yaksh, 1993). The anti-allodynic effect of these two peptides parallels their anti-nociceptive effect with similar ED50. We have previously found that the order of potency for the various effects of i.t. morphine is the following: antinociception in normal rats . anti-nociception in allodynic rats . anti-allodynia (Yu et al., 1997a), indicating that the
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spinal sensitivity to morphine is reduced in spinally injured rats and allodynia is relatively insensitive to morphine treatment. It is unclear why such difference is not observed with more selective opioid receptor agonists. It should be noted, however, that the dose-response curve for both DAMGO and DPDPE are rather steep, making the estimation of ED50 difficult in the present study. Both pre- and post-synaptic mechanisms have been proposed for the spinal anti-nociceptive effect of opioids (Yaksh, 1987). The selectivity of opioids in suppressing nociceptive input has been suggested to be derived primarily from a pre-synaptic action (Yaksh, 1987, 1991). The mechanical allodynia-like behavior in our model is likely to be triggered by activation of low threshold afferents since it was evoked by very low intensity stimuli and was not abolished by resiniferatoxin, an ultrapotent capsaicin analogue that abolishes responses mediated by unmyelinated afferents (Hao et al., 1996). We have suggested therefore that the anti-allodynic effect of i.t. opioids, at least against mechanical stimuli, is through post-synaptic action (Yu et al., 1997a). Although the majority of the spinal m-and dopioid receptors are located pre-synaptically, a substantial number of post-synaptic receptors are found (Besse et al., 1990; Gouarde´res et al., 1991; Arvidsson et al., 1995; Stevens and Seybold, 1995). Furthermore, a recent study using in situ hydridization has demonstrated the presence of mand d-opioid receptors in large to medium sized dorsal root ganglion cells, indicating that the pre-synaptic action of mand d-opioid receptor agonists may not be so selective on Cafferent input (Maekawa et al., 1994). Unlike the mechanical allodynia, the cold allodynia was abolished by resiniferatoxin treatment, indicating that it is
Fig. 6. Effects of cumulative doses of i.t. U50488H on responses of rats to cold (A) and tactile (B) stimulation. The Wilcoxon signed-ranks test indicated that DAMGO caused significant increase in cold and tactile response starting at 100 ng i.t. (P , 0.05).
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mediated by capsaicin-sensitive afferents (Hao et al., 1996). We however did not observe any marked difference in the potency of i.t. DAMGO and DPDPE in alleviating these two components of the allodynia-like behaviors. This may indicate that stimulation intensity cannot be matched between these two modalities as previous studies have established that the potency of spinal opioid anti-nociception is influenced by stimulus intensity (Saeki and Yaksh, 1993). Furthermore, the assessment of the two responses is also different, making it difficult to make direct comparisons. 4.2. Effects of the k-opioid receptor agonist U50488H on allodynia-like behaviors In contrast to the effect of m- and d-opioid agonists, the kopioid receptor agonist U50488H did not elicit thermal antinociception. Similar results have been reported in other studies and it has been shown that the spinal anti-nociceptive effect of k-opioid receptor agonists depends on the test used and stimulus intensity (Schmauss and Yaksh, 1984; Millan, 1989). Intrathecal U50488H did not alleviate the chronic allodynia-like behavior, but instead enhanced the allodynia. Thus, U50488H further decreased the vocalization threshold of the rats to von Frey hair stimulation, increased the cold response and also triggered and enhanced tactile allodynia. It is unlikely that the pro-allodynic action of U50488H was due to a non-specific excitatory action as we have observed similar effects with CI-977, another high affinity k-opioid receptor agonist (Hao et al., unpublished observations). Intrathecal dynorphin and related peptides have complex effects on spinal cord function, including excitation, depression and neurotoxicity. Although some of the spinal effects of dynorphin are non-opioid in nature (Stewart and Isaac, 1991; Vanderah et al., 1996), activation of spinal k-opioid receptors has been reported to increase the excitability of dorsal horn neurons (Knox and Dickenson, 1987; Hylden et al., 1991). Moreover, it has been reported by numerous laboratories that spinal administration of dynorphin and U50488H antagonized anti-nociception induced by morphine or other m-opioid receptor agonists (Dickenson and Knox, 1987; Fujimoto et al., 1990; Song and Takemori, 1991). In the present study, we also observed that the antiallodynic effect of DAMGO was markedly reduced by U50488H, indicating the anti-analgesic action of k-opioid receptor agonists in the spinally injured rats. Such m-opioid receptor blocking effect of U50488H may account for the enhancing effect on the allodynia-like behavior as naloxone exerted similar effects (Xu et al., 1994). Drugs with k-opioid receptor agonist properties have become available for human use (Hammond, 1991) and it has been suggested that they may represent novel analgesics devoid of other side effects associated with opioids (Millan, 1990). Our results, however, suggest this is unlikely to be the case for central pain after spinal cord injury. Moreover, caution needs to be exercised in administering k-opioid
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receptor agonists in such patients as they may enhance or trigger their pain. 4.3. Clinical implications Central pain remains difficult to manage with conventional approaches (Boivie, 1994). In two clinical studies, Glynn et al. (1986) and Glynn et al. (1988) reported that i.t. morphine alleviated central pain in some patients with multiple sclerosis or painful arachnoiditis. We have previously shown that i.t. morphine produced full alleviation of the mechanical allodynia in spinally injured rats, albeit with reduced potency compared to its anti-nociceptive effect (Yu et al., 1997a). In the present study, we showed that i.t. administration of selective agonists of m- and dopioid receptors are also effective in treating central painrelated behavior in spinally injured rats, suggesting that not only morphine, but also receptor selective opioid agonists may be clinically useful in the management of chronic central pain of spinal origin. Spinal administration of morphine is associated with numerous side effects and tolerance develops rapidly to its anti-allodynic effect (Yu et al., 1997b). Receptor selective opioids may have several advantages over morphine. First, fewer side effects may be associated with selective agonists of opioid receptor subtypes. Second, receptor selective opioids may produce analgesia in patients tolerant to agonists of another type of opioid receptor. Finally, alternative application of m- and d-opioid receptor agonists may also delay the development of tolerance. These exciting possibilities, however, remain to be tested clinically as no d receptor selective agonists are available for human use. It also needs to be emphasized that the model we used is a model of central pain with injury at the spinal level. Thus, the pain developed is likely to have a spinal mechanism. Consequently, it is possible that central pain after brain injury may require different treatments.
Acknowledgements This study was supported by Swedish Medical Research Council (07913 and 12168), Astra Pain Control AB, the Biomed II programme of the European Commission, Kapten Arthur Eriksson Foundation, the Swedish Medical Association and research fund of the Karolinska Institute.
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Treatment of chronic allodynia in spinally injured rats: effects of intrathecal selective opioid receptor agonists Jing-Xia Hao, Wei Yu, Zsuzsanna Wiesenfeld-Hallin, Xiao-Jun Xu* Department of Medical Laboratory Sciences and Technology, Division of Clinical Neurophysiology, Karolinska Institute, Huddinge University Hospital, S-141 86 Huddinge, Sweden Received 26 August 1997; received in revised form 20 November 1997; accepted 4 December 1997
Abstract We examined the effects of intrathecal (i.t.) selective opioid receptor agonists in alleviating mechanical and cold allodynia in spinally injured rats. Both DAMGO ([D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin, a m-opioid receptor agonist) and DPDPE ([D-Phe2,D-Phe5]-enkephalin, a d-opioid receptor agonist) dose-dependently relieved the chronic allodynia-like behavior at doses selective for their respective receptors. The anti-allodynic effect of DAMGO and DPDPE was reversed by the selective m- and d-opioid receptor antagonists CTOP (DPhe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2) and naltrindole, respectively. In contrast, the selective k-opioid receptor agonist U50488H did not alleviate the allodynia-like behavior, but rather enhanced it. The anti-nociceptive and anti-allodynic effect of i.t. DAMGO was blocked by U50488H. Thus, activation of spinal m- and d-, but not k-opioid receptors produced anti-allodynic effect in this model of central pain. Drugs which act selectively on opioid receptor subtypes may be useful in managing chronic central pain of spinal cord origin. 1998 International Association for the Study of Pain. Published by Elsevier Science B.V. Keywords: Chronic pain; Intrathecal; Morphine; Neuropathic pain; Opioids; Spinal cord injury
1. Introduction Neuropathic pain, which can occur after nervous system injury, is usually difficult to treat. This is particularly true for central pain after injury or dysfunction to the central nervous system (CNS) (Tasker, 1990; Boivie, 1994). The successful development of suitable animal models for these human pain conditions will not only be useful in elucidating the mechanisms of these painful states, but also in exploring effective treatments. Several animal models for neuropathic pain following peripheral nerve injury are available which have generated a large body of literature on the pharmacological treatment of the abnormal painrelated behaviors in these models (Bennett, 1993; Zeltser and Seltzer, 1994). We have recently developed a rat model of chronic pain-
* Corresponding author. Division of Clinical Neurophysiology, Huddinge University Hospital, S-141 86 Huddinge, Sweden. Tel.: +46 8 58582213; fax: +46 8 7748856; e-mail: [email protected]
related behaviors after ischemic spinal cord injury which in many aspects mimics central pain in spinally injured patients. Thus, we have observed marked mechanical and cold allodynia-like behaviors in spinally injured rats in the dermatomes at or rostral to the injured spinal segments (Xu et al., 1992a, 1994). Furthermore, animals also exhibited autotomy and ex-cessive scratching, behaviors that may indicate the presence of spontaneous pain or dysesthesia (Xu et al., 1992a). We and others have suggested that this may represent an animal model for central pain of spinal origin which provides a valuable tool to study the mechanisms and treatments of this particular type of refractory painful condition (Yezierski, 1996; Wiesenfeld-Hallin et al., 1997). We have conducted systematic studies examining the pharmacological management of chronic allodynia-like behaviors in these spinally injured rats. Similar to some clinical reports, we found that systemically administered local anesthetics, tocainide and mexiletine, were effective in alleviating the chronic allodynia-like behavior (Xu et al., 1992a,b). Furthermore, the allodynia could be relieved by
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systemic administration of antagonists of N-methyl-daspartate (NMDA) receptors, nitric oxide synthase inhibitors and antagonists of CCKB receptors (Xu et al., 1994; Hao and Xu, 1996a,b), indicating the possible involvement of these transmitter systems in the generation and maintenance of the chronic allodynia-like state. We are particularly interested in exploring the anti-allodynic efficacy of morphine in our model of central pain as it is a controversial issue in clinic practice whether or not opiates are effective in treating neuropathic pain (Arne´r and Meyerson, 1988; Arne´r and Meyerson, 1993; Portenoy et al., 1990; McQuay et al., 1992). Moreover, the efficacy of morphine in treating central pain has not been thoroughly evaluated (Hammond, 1991), although the general impression is that morphine is rather ineffective (Boivie, 1994; Bowsher and Nurmikko, 1996). We have evaluated the efficacy and potency of morphine in alleviating the allodynialike behaviors in this model after systemic, intrathecal (i.t.) and intracerebroventricular (i.c.v.) administration (Yu et al., 1997a). Systemic morphine was ineffective in alleviating allodynia at sub-sedative doses. Similarly, i.c.v. morphine only reduced the vocalization component of the allodynic responses. In contrast, i.t. morphine dose-dependently alleviated allodynia without causing sedation, although the doses of morphine required to produce the anti-allodynic effect were higher than those for anti-nociception. These results suggest that spinal administration of morphine may be useful in treating central pain in patients with spinal cord injury (Yu et al., 1997a). Three major receptor subtypes, m, d and k, mediate the actions of opioids in the nervous system (Martin et al., 1976; Yaksh, 1987; Mansour et al., 1988). Agonists of all three opioid receptors induce anti-nociception at the spinal level (Yaksh, 1987), although the effects of k-opioid receptor agonists are less clear (Schmauss and Yaksh, 1984; Knox and Dickenson, 1987; Millan, 1989; Hylden et al., 1991). It has also been reported that spinal administration of k-opioid receptor agonists antagonized the effects of morphine and other m-opioid receptor agonists (Dickenson and Knox, 1987; Fujimoto et al., 1990; Song and Takemori, 1991). The present studies were undertaken to explore the effects of i.t. administration of selective agonists for m-, d- and k-opioid receptors on the mechanical and cold allodynia-like behaviors in spinally injured rats. The selective antagonists CTOP (Kramer et al., 1989) and naltrindole (Portoghese et al., 1988) were used to further confirm the receptor subtypes involved. The interactions between the k-opioid receptor agonist U50488H and mopioid receptor agonist DAMGO were also examined.
2. Materials and methods 2.1. Photochemically-induced spinal cord ischemia The subjects were female Sprague–Dawley rats (B & K Universal, Stockholm, Sweden). All experiments were
approved by the local research ethics committee. The method for inducing photochemical spinal cord lesion has been described in detail previously (Xu et al., 1992a). Briefly, the rats were anesthetized with chloral hydrate (Sigma; 300 mg/kg, i.p.) and one jugular vein was catheterized. Vertebrae T11–L2 were exposed after a midline incision of the skin on the back. The animals were positioned beneath a tuneable argon ion laser (Innova, Model 70, Coherent Laser Products Division) and irradiated with a knife edge beam, which was used to cover the T13 vertebra, with an average power of 0.16 W for 10 min. No laminectomy was performed. Immediately before and 5 min after the start of the irradiation, erythrosin B (Red No. 3, AldrichChemie) was injected intravenously in 0.9% saline at a dose of 32.5 mg/kg. After irradiation the incision was closed and the animals were kept warm for 2 h. Their bladders were emptied manually 2–3 times a day until normal function was regained. 2.2. Test of responses to mechanical and cold stimuli A set of calibrated von Frey hairs was used to test the vocalization thresholds of the rats to graded mechanical touch/pressure ranging from 0.021 to 410 g. During testing the rats were gently restrained in a standing position and the von Frey hair was pushed onto the skin until the filament became bent. The frequency of the stimulation was about 1 Hz and at each intensity, the stimuli were applied 5–10 times. The intensity of stimulation which induced consistent vocalization (.75% response rate) was considered as pain threshold. Although a number of reactions were noted in response to light mechanical stimulation in allodynic rats after spinal cord lesion (such as biting, escaping and hindlimb flexion), vocalization was chosen to represent the pain-like response as it was most consistent and easiest to characterize. The responses of rats to tactile stimulation was tested with the blunt point of a pencil gently brushing the skin of the rats in a rostral-caudal direction. The frequency of the stimulation was about 1 Hz and responses were graded with a score of 0, no observable response; 1, transient vocalization and moderate effort to avoid probe; 2, consistent vocalization and aversive reactions; and 3, sustained and prolonged vocalizations, aggressive behaviors. Normal rats exhibit no reactions to such tactile stimuli (score 0). The response of the rats to cold stimuli was evaluated by spraying ethyl chloride (Medikema AB, Perstorp, Sweden) on the shaved skin area exhibiting mechanical allodynia. The response of the rat was graded with a score of 0, no observable response; 1, localized response (skin twitch and contraction), no vocalization; 2, transient vocalization, moderate struggle; and 3, sustained vocalization and aversive reactions. The spray produced an intense cold sensation when applied to the experimenter’s forearm and skin temperature was reduced to around 0°C immediately after application, with rapid recovery.
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The light beam from a projector bulb was focused 1–2 cm from the tip of the tail and the latency of the tail flick was measured. The intensity of the thermal stimulus was adjusted so that the baseline latency was at 3–4 s and a 10 s cut off time was imposed to avoid tissue damage.
groups. The data are expressed as medians ± MAD (median derived absolute value) or as mean ± SEM and were analysed with the Wilcoxon signed-ranks test. In order to estimate ED50 and compare the anti-allodynic and antinociceptive potency of DAMGO and DPDPE, we have also calculated the % maximal possible effect (MPE) based on the formula:
2.4. Implantation of i.t. catheters
%MPE = (post − drug response − baseline=
2.3. Tail flick test
The i.t. catheter was implanted according to a modification of the technique described by Storkson et al. (1996). After anesthesia with methohexital (Brietal, Lilly, IN, USA), a catheter (PE 10, o.d. 0.61 mm) was inserted into the subarachnoid space through a guide cannula connected to a 20 gauge needle which punctured the dura at the level of the cauda equina. The catheter was then carefully implanted rostrally, aiming its tip at the lumbar enlargement. The proper location of the catheter was examined before the actual pharmacological experiments by assessing sensory and motor blockade after i.t. injection of 7 ml lidocaine (50 mg/ml; Astra, So¨derta¨lje, Sweden) and was confirmed after the experiments by laminectomy. 2.5. Experimental design Thirteen chronic allodynic rats were randomly assigned to three groups and DAMGO (n = 6), DPDPE (n = 4) or U50488H (n = 3) were administered i.t. in a cumulative dose regime at 20 min intervals. The von Frey hair thresholds and the responses of the rats to tactile and cold stimulation were assessed before and every 10 min after i.t. administration. The tail flick latency was only tested once after each dose at 10 min in order to avoid tissue damage. The rats that received DAMGO as their first injection were used again 1 week later where DPDPE (n = 4) or U50488H (n = 2) were administered. No differences in the effects of DPDPE and U50488 were noted between rats that did or did not receive DAMGO previously and thus results from these two experiments were combined for data presentation and analysis. 2.6. Drugs and statistics DAMGO [(D-Ala2,N-Me-Phe4,Gly5-ol)-enkephalin], DPDPE [(D-Phe2,D-Phe5)-enkephalin] and CTOP (D-PheCys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2) were obtained from Peninsula Laboratories (Merseyside, UK). U50488H (trans-(±)-3,4-dichloro-N-methyl-N-[2-(1-pyrolidinyl)cyclohexnoyl]benzeneacetamide methanesulphonate) and naltrindole (17-cyclopropylmethyl-6,7-dehydro-4,5a-epoxy-3,14-dihydro-6,7-2′,3′-indolomorphinan) were from Research Biochem (Natick, MA, USA). All drugs were dissolved in saline and injected i.t. in a volume of 10 ml followed by 15 ml saline to flush the catheter. Rats were randomly assigned to the different treatment
maximal response − baseline) × 100 where maximal response is 10 s for the tail flick test and 73 g (response threshold in normal rats) in the von Frey hair test, respectively. Linear regression was used to examine the dose-dependent effect of DAMGO and DPDPE and to calculate ED50.
3. Results 3.1. Allodynia-like behaviors in spinally injured rats Two weeks to 3 months after the ischemic injury, mechanical and cold allodynia-like responses were observed in about 40% of the rats (Xu et al., 1992a; Hao and Xu, 1996a; Hao and Xu, 1996b). The vocalization threshold to pressure exerted by von Frey hairs in normal rats was 73– 95 g and there were no reactions to subthreshold stimuli. In contrast, there was a marked decrease in vocalization threshold to mechanical pressure (median 0.94 g, range 0.18–7.1 g) in the animals with pain-related behavior. Other behaviors, such as skin twitch, jumping and escaping was also evoked by low intensity mechanical touch/pressure. Light tactile stimulation triggered painful reactions in some, but not all, rats where low intensity mechanical touch/pressure generated painful reactions. In response to the cold spray, normal rats exhibited no or local responses (score 0 or 1). In contrast, the allodynic rats exhibited clearcut aversive reactions, including vocalization, licking or scratching of the stimulated area, avoidance and escaping. Mechanical and cold allodynia-like behaviors were usually found in the same animal. The skin areas where increased response to mechanical and cold stimulation could be observed corresponded to one or more dermatomes at the rostral edge of the lesioned spinal segment. As the epicenter of the lesion was near the lumbar enlargement, the allodynic area was usually at the mid or lower back and abdomen. In the majority of rats the symptoms lasted for months without signs of remission. The pain-like behaviors occurred only when the rats were tested and the animals otherwise exhibited no overt signs of discomfort. Rats that were included in the present study were irradiated 3–8 months prior to the pharmacological experiments, and all exhibited robust allodynia-like behavior to mechanical and cold stimuli for at least 1 month. Some rats
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also exhibited tactile allodynia. Some motor deficits were observed in these rats, which were mainly expressed as minor deficits in walking, slowed righting reflexes and reduced performance on the inclined plane test. 3.2. Effect of DAMGO The smallest dose of DAMGO tested (1 ng) produced no significant effect on vocalization threshold to von Frey hair stimulation (Fig. 1A), tail flick latency (Fig. 1B) and cold responses (Fig. 2A). A cumulative dose of 10 ng DAMGO increased the vocalization threshold of four out of six rats to stimulation with von Frey hairs. In fact the response thresh-
old was the same as in normal animals (73 g) (Fig. 1A). The responses of five out of six rats to cold was also normalized (Fig. 2A). The tail flick latency was also significantly prolonged by DAMGO at this dose (Fig. 1B). At a 30 ng cumulative dose, DAMGO normalized the vocalization threshold of all rats (Fig. 1A) and the tail flick latency reached cut-off value in five out of six rats (Fig. 1B). Interestingly, at this dose, the response of all rats to cold was abolished (score 0) which is below normal response. No further increase in vocalization threshold to stimulation with von Frey hairs was observed after a 100 ng cumulative dose of DAMGO, with only two out of six rats showing a response threshold (103 g) that was somewhat higher than normal values (Fig. 1A). Four of the six rats also exhibited tactile allodynia (Fig. 2B). These responses were also dose-dependently reduced by i.t. DAMGO in a fashion similar to the changes in the vocalization threshold to stimulation with von Frey hairs (Fig. 2B). In three rats, the duration of action of 100 ng i.t. DAMGO was followed and its anti-allodynic and anti-nociceptive effect was maintained for 45 min, partially recovered at 60 min and fully reversed at 90 min. In another three rats, the selective m-opioid receptor antagonist CTOP (10 or 30 ng) was administered i.t. 20 min after a cumulative dose of 100 ng DAMGO and a rapid reversal of the anti-allodynic and anti-nociceptive effect of DAMGO was observed. 3.3. Effect of DPDPE
Fig. 1. Effects of cumulative doses of intrathecal (i.t.) DAMGO on vocalization threshold to mechanical touch/pressure (A) and tail flick latency (B) in allodynic rats (n = 6). Note the logarithmic scale on the x-axis in both (A) and (B) and on the y-axis in (A). In this figure and Figs. 3 and 5, data are expressed as median ± MAD for vocalization threshold and as mean ± SEM for tail flick latency. DAMGO caused significant increase in vocalization threshold and tail flick latency starting from a 10 ng cumulative i.t. dose (*P , 0.05, compared to control value with Wilcoxon signed-ranks test).
Intrathecal DPDPE at 1 mg produced marked increase in vocalization threshold to von Frey hair stimulation in four out of eight rats. However, in the remaining four rats, there was little or no effect (Fig. 3A). DPDPE did not produce a significant effect on the response to cold at 1 mg (Fig. 4A). Increasing the dose of DPDPE to 3 mg markedly increases the vocalization threshold in all rats tested with normalization observed in five out of eight rats (Fig. 3A). The response to cold was also significantly reduced at this dose (Fig. 4A). At a cumulative dose of 10 mg DPDPE, the vocalization threshold to von Frey hairs and cold responses were normalized in all rats (Figs. 3A and 4A). No further increase in vocalization threshold or decrease in cold response was noted after a cumulative dose of 30 mg DPDPE (Figs. 3A and 4A). Four of the eight rats tested also exhibited tactile allodynia. These responses were dose-dependently reduced by i.t. DPDPE in a fashion similar to changes in vocalization threshold to von Frey hairs (Fig. 4B). The tail flick latency was also dose-dependently prolonged by i.t. DPDPE with a significant effect already observed at 1 mg (Fig. 3B). In four rats, the duration of action of a cumulative dose of 30 mg DPDPE was followed and was found to last between 30 and 45 min. In another four rats, the selective d-opioid receptor antagonist naltrindole (10 or 30 mg) was administered i.t. 10 min after 30 mg i.t. DPDPE and rapid reversal of
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threshold to von Frey hair stimulation was noted after a 100 ng cumulative dose of U50488H (Fig. 5A). Increasing the dose of U50488H to 1 or 10 mg still did not produce an anti-allodynic effect (Fig. 5A). Intrathecal U50488H also triggered tactile allodynia in rats which did not exhibit such behavior before the experiments and enhanced the already existing tactile allodynia (Fig. 6B). Intrathecal U50488H did not significantly alter the tail flick latency (Fig. 5B). 3.6. Effect of U50488H on the anti-allodynic effect of DAMGO One hundred nanograms DAMGO was administered i.t. 20 min after a 10 mg cumulative dose of U50488H in four rats. In contrast to the effectiveness of this large dose of DAMGO in suppressing the allodynia-like behavior in rats
Fig. 2. Effects of cumulative doses of i.t. DAMGO on responses of rats to cold (A) and tactile (B) stimulation. In this figure and Figs. 4 and 6, the number of rats showing different response scores are illustrated and the scores are represented as open columns (score 0); hatched columns (score 1), densely hatched columns (score 2) and dotted columns (score 3) (see Section 2 for detailed description of scoring criteria). The Wilcoxon signed-ranks test indicated that DAMGO caused significant reduction in the cold response starting at 10 ng (P , 0.05) and in the tactile response starting at 30 ng (P , 0.05).
the anti-allodynic and anti-nociceptive effect of the agonist was observed. 3.4. Comparison of the potency for the anti-allodynic and anti-nociceptive effect of DAMGO and DPDPE In order to compare the potency of DAMGO and DPDPE on reactions to different types of stimuli, the responses to von Frey hairs and tail flick test were converted to % change. Regression analysis indicated that both DAMGO and DPDPE caused significant dose-dependent anti-allodynic and anti-nociceptive effects. The ED50 for the anti-allodynic and anti-nociceptive effects of DAMGO were 5.8 and 12.7 ng, respectively and for DPDPE 0.53 and 0.34 mg, respectively. 3.5. Effect of U50488H U50488H administered i.t. at 1 or 10 ng did not significantly alter vocalization threshold to mechanical pressure and cold responses (Figs. 5A and 6A). However, at a 30 ng cumulative dose, there was a significant decrease in vocalization threshold to von Frey hair stimulation (Fig. 5A). There was also an increase in the cold responses after 30 ng U50488H (Fig. 6A). Further reduction in vocalization
Fig. 3. Effects of cumulative doses of i.t. DPDPE on vocalization threshold to mechanical touch/pressure (A) and tail flick latency (B) in allodynic rats (n = 8). Note the logarithmic scale on the x-axis in both (A) and (B) and on the y-axis in (A). DPDPE cause a significant dose-dependent increase in vocalization threshold and tail flick latency (*P , 0.05, compared to control value with Wilcoxon signed-ranks test).
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which did not receive U50488H previously, three of four rats failed to respond to DAMGO and only a moderate increase in vocalization threshold was observed in one rat. The anti-nociceptive effect of DAMGO was also reduced by U50488H.
4. Discussion 4.1. Anti-allodynic effect of m- and d-opioid receptor agonists In the present study, we observed that i.t. administration of selective agonists of m- or d-opioid receptors dose-dependently alleviated the mechanical allodynia-like behavior in spinally injured rats. The doses of these two agonists employed were comparable to or lower than those used in previous studies (Drower et al., 1991; Miaskowski et al.,
Fig. 5. Effects of cumulative doses of i.t. U50488H on vocalization threshold to mechanical touch/pressure (A) and tail flick latency (B) in allodynic rats (n = 5). Note the logarithmic scale on the x-axis in both (A) and (B) and on the y-axis in (A). U50488H caused a significant decrease in vocalization threshold (*P , 0.05, compared to control value with Wilcoxon signed-ranks test). No significant effect on tail flick latency was observed.
Fig. 4. Effects of cumulative doses of i.t. DPDPE on the responses of rats to cold (A) and tactile (B) stimulation. Wilcoxon signed-ranks test indicated that DAMGO caused significant reduction in cold response starting at 3 mg (P , 0.05). No significant effect on tactile response was observed due to the low number of rats exhibiting such behavior before DPDPE administration.
1991; Malmberg and Yaksh, 1992; Tiseo and Yaksh, 1993; Guirimand et al., 1994). The effects of the agonists were reversed by respective antagonists, indicating receptor mediation of the anti-allodynic effects of the m- and d-opioid agonists. No noticeable side effects, such as sedation or motor deficits, were observed after even the highest doses of these two opioid receptor agonists. In agreement with previous studies, i.t. DAMGO or DPDPE caused potent thermal anti-nociception (Drower et al., 1991; Malmberg and Yaksh, 1992; Tiseo and Yaksh, 1993). The anti-allodynic effect of these two peptides parallels their anti-nociceptive effect with similar ED50. We have previously found that the order of potency for the various effects of i.t. morphine is the following: antinociception in normal rats . anti-nociception in allodynic rats . anti-allodynia (Yu et al., 1997a), indicating that the
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spinal sensitivity to morphine is reduced in spinally injured rats and allodynia is relatively insensitive to morphine treatment. It is unclear why such difference is not observed with more selective opioid receptor agonists. It should be noted, however, that the dose-response curve for both DAMGO and DPDPE are rather steep, making the estimation of ED50 difficult in the present study. Both pre- and post-synaptic mechanisms have been proposed for the spinal anti-nociceptive effect of opioids (Yaksh, 1987). The selectivity of opioids in suppressing nociceptive input has been suggested to be derived primarily from a pre-synaptic action (Yaksh, 1987, 1991). The mechanical allodynia-like behavior in our model is likely to be triggered by activation of low threshold afferents since it was evoked by very low intensity stimuli and was not abolished by resiniferatoxin, an ultrapotent capsaicin analogue that abolishes responses mediated by unmyelinated afferents (Hao et al., 1996). We have suggested therefore that the anti-allodynic effect of i.t. opioids, at least against mechanical stimuli, is through post-synaptic action (Yu et al., 1997a). Although the majority of the spinal m-and dopioid receptors are located pre-synaptically, a substantial number of post-synaptic receptors are found (Besse et al., 1990; Gouarde´res et al., 1991; Arvidsson et al., 1995; Stevens and Seybold, 1995). Furthermore, a recent study using in situ hydridization has demonstrated the presence of mand d-opioid receptors in large to medium sized dorsal root ganglion cells, indicating that the pre-synaptic action of mand d-opioid receptor agonists may not be so selective on Cafferent input (Maekawa et al., 1994). Unlike the mechanical allodynia, the cold allodynia was abolished by resiniferatoxin treatment, indicating that it is
Fig. 6. Effects of cumulative doses of i.t. U50488H on responses of rats to cold (A) and tactile (B) stimulation. The Wilcoxon signed-ranks test indicated that DAMGO caused significant increase in cold and tactile response starting at 100 ng i.t. (P , 0.05).
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mediated by capsaicin-sensitive afferents (Hao et al., 1996). We however did not observe any marked difference in the potency of i.t. DAMGO and DPDPE in alleviating these two components of the allodynia-like behaviors. This may indicate that stimulation intensity cannot be matched between these two modalities as previous studies have established that the potency of spinal opioid anti-nociception is influenced by stimulus intensity (Saeki and Yaksh, 1993). Furthermore, the assessment of the two responses is also different, making it difficult to make direct comparisons. 4.2. Effects of the k-opioid receptor agonist U50488H on allodynia-like behaviors In contrast to the effect of m- and d-opioid agonists, the kopioid receptor agonist U50488H did not elicit thermal antinociception. Similar results have been reported in other studies and it has been shown that the spinal anti-nociceptive effect of k-opioid receptor agonists depends on the test used and stimulus intensity (Schmauss and Yaksh, 1984; Millan, 1989). Intrathecal U50488H did not alleviate the chronic allodynia-like behavior, but instead enhanced the allodynia. Thus, U50488H further decreased the vocalization threshold of the rats to von Frey hair stimulation, increased the cold response and also triggered and enhanced tactile allodynia. It is unlikely that the pro-allodynic action of U50488H was due to a non-specific excitatory action as we have observed similar effects with CI-977, another high affinity k-opioid receptor agonist (Hao et al., unpublished observations). Intrathecal dynorphin and related peptides have complex effects on spinal cord function, including excitation, depression and neurotoxicity. Although some of the spinal effects of dynorphin are non-opioid in nature (Stewart and Isaac, 1991; Vanderah et al., 1996), activation of spinal k-opioid receptors has been reported to increase the excitability of dorsal horn neurons (Knox and Dickenson, 1987; Hylden et al., 1991). Moreover, it has been reported by numerous laboratories that spinal administration of dynorphin and U50488H antagonized anti-nociception induced by morphine or other m-opioid receptor agonists (Dickenson and Knox, 1987; Fujimoto et al., 1990; Song and Takemori, 1991). In the present study, we also observed that the antiallodynic effect of DAMGO was markedly reduced by U50488H, indicating the anti-analgesic action of k-opioid receptor agonists in the spinally injured rats. Such m-opioid receptor blocking effect of U50488H may account for the enhancing effect on the allodynia-like behavior as naloxone exerted similar effects (Xu et al., 1994). Drugs with k-opioid receptor agonist properties have become available for human use (Hammond, 1991) and it has been suggested that they may represent novel analgesics devoid of other side effects associated with opioids (Millan, 1990). Our results, however, suggest this is unlikely to be the case for central pain after spinal cord injury. Moreover, caution needs to be exercised in administering k-opioid
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receptor agonists in such patients as they may enhance or trigger their pain. 4.3. Clinical implications Central pain remains difficult to manage with conventional approaches (Boivie, 1994). In two clinical studies, Glynn et al. (1986) and Glynn et al. (1988) reported that i.t. morphine alleviated central pain in some patients with multiple sclerosis or painful arachnoiditis. We have previously shown that i.t. morphine produced full alleviation of the mechanical allodynia in spinally injured rats, albeit with reduced potency compared to its anti-nociceptive effect (Yu et al., 1997a). In the present study, we showed that i.t. administration of selective agonists of m- and dopioid receptors are also effective in treating central painrelated behavior in spinally injured rats, suggesting that not only morphine, but also receptor selective opioid agonists may be clinically useful in the management of chronic central pain of spinal origin. Spinal administration of morphine is associated with numerous side effects and tolerance develops rapidly to its anti-allodynic effect (Yu et al., 1997b). Receptor selective opioids may have several advantages over morphine. First, fewer side effects may be associated with selective agonists of opioid receptor subtypes. Second, receptor selective opioids may produce analgesia in patients tolerant to agonists of another type of opioid receptor. Finally, alternative application of m- and d-opioid receptor agonists may also delay the development of tolerance. These exciting possibilities, however, remain to be tested clinically as no d receptor selective agonists are available for human use. It also needs to be emphasized that the model we used is a model of central pain with injury at the spinal level. Thus, the pain developed is likely to have a spinal mechanism. Consequently, it is possible that central pain after brain injury may require different treatments.
Acknowledgements This study was supported by Swedish Medical Research Council (07913 and 12168), Astra Pain Control AB, the Biomed II programme of the European Commission, Kapten Arthur Eriksson Foundation, the Swedish Medical Association and research fund of the Karolinska Institute.
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